The rTS gene, previously discovered in our laboratory, produces a naturally occurring antisense RNA (rTSalpha) to human thymidylate synthase (TS) RNAs and two proteins (rTSalpha and rTSbeta). Since the initial discovery that the rTS gene produces protein products as well as antisense RNA, this laboratory has endeavored to identify the biologic functions of the proteins. In gram- negative bacteria, many functions (e.g. bioluminescence) are activated when the bacteria reach high cell densities in a process called quorum sensing. Quorum sensing depends upon the secretion and sensing of species-specific nonpolar signal molecules (acyl homoserine lactones, acyl-HSLs) that arise from the metabolism of methionine. It is becoming evident that this process may be a universal communication mechanism for regulating a variety of microbial gene functions that vary with cell density. Preliminary data in this application provide evidence that rTSbeta over-expressing cells regulate TS levels in a cell density dependent manner. This regulation is controlled by the over-production of intercellular signal molecules compared with cells that do not over-produce rTSbeta. Data further suggest these molecules are novel nonpolar metabolites of methionine, likely to be similar in chemical structure to microbial quorum sensing molecules. We hypothesize the rTS is a growth suppressing gene that regulates cell growth by instigating the decline of TS protein and RNA through a newly discovered signalling pathway that is the subject of this application. Since TS is a major target for drug design and cancer chemotherapy, and rTSbeta is expressed in tissues of the human gut, this pathway may be a prime candidate for drug development and for modulating tumor response to therapy. We propose to investigate these possibilities in three Specific Aims: 1) Study the mechanism for down-regulation of TS in trans by rTSbeta over- expressing cells. Identification of other cell population density regulated genes. 2) Examine the relation of rTSbeta over-expression to Met metabolism. Signal molecule isolation and characterization. 3) Develop an in vitro assay for acyl-HSL synthesis. Qualification of the role of rTS proteins and other participants in this reaction. Purification of rTS enzyme and kinetic analysis of the acyl-HSL biosynthetic reaction.